The overall objective of this study is to understand the molecular mechanisms underlying the function of ionic channels. In order to circumvent the difficulties of carrying out biochemical studies in vivo, studies will use less complex in vitro systems. The first part of this study will be concerned with improving the methodology used to study voltage-dependent channels in reconstituted vesicles and membrane fragments. Membrane potentials will be established in populations of small phospholipid vesicles either by K ion gradients in the presence of valinomycin, or by the light-induced electrogenic proton flux of bateriorhodopsin. Next, ionic fluxes across reconstituted vesicles containing Na channels will be studied. Membrane fractions from the innervated membrane of the electroplax of Electrophorus electricus are incorporated into phospholipid vesicles. By combining the techniques to control the membrane potential across small vesicles with the use of specific pharmacological tools we will study the ionic fluxes through these Na channels. These fluxes and the binding of Saxitoxin will serve as assays of the functional state of the channels. We will study the conditions required for long term survival of the channel in vitro and for its solubilization and reinsertion into lipid vesicles. Finally, we will improve and extend the use of a recently developed artifical membrane system, cell-size phospholipid vesicles. This system is devoid of organic solvent and is amenable to study with electrophysiological, chemical and optical techniques. Further development of this system will emphasize the reconstitution of membrane proteins, especially ionic chennels in these vesicles. Three channels which have been maintained in vitro will be used: the voltage-dependent anion selective channel from mitochondria, porine from the external membrane of E. coli and acetylcholine receptors.